How to formulate peptides into metered dose inhalers (MDI) is a bottleneck issue hampering the clinical translation of relevant products. In our previous studies, a bottom-up method to prepare peptide-containing particles for MDI was reported. Nevertheless, the formation mechanism of the particles remains unclear. In this work, considering the production workflow, a confinement-segregation theory was put forward as a hypothesis to explain the formation mechanism. Confinement and segregation were two major processes during formation, and the definitions were provided in detail. Based on the theory, some factors influencing particle formation were also discussed, which promoted the future formulation design. It is believed that the proposed theory will provide new insights into the study of peptide-containing MDI, and boost the clinical translation.